Immunological diagnostics utilizing polystyrene latex particles of 0.15 to 0.25 micron



United States Patent IMMUNOLOGICAL DTAGNGSTICS UTILIZING POLYSTYRENELATEX PARTICLES 0F 0.15

TO 0.25 MICRON Roy T. Fisk, Glendale, Calif, assignor to HylandLaboratories No Drawing. Filed Oct. 27, 1959, Ser. No. 848,922 7 Claims.(Cl. 167-84.5)

This invention relates to diagnostic procedures for determining thepresence or absence in blood and other body fluids of various substanceswhich indicate an abnormal or pathological state or condition. Morespecifically, this invention is concerned with novel diagnostic reagentsfor immunological analysis of body substances.

A number of pathological states or conditions in both human beings andanimals can be, and often are, diagnosed through the application ofwell-known immunological principles. It will be understood that theword, animals when jemployed herein includes human beings, unless thereis a specific statement to the contrary. Animals which are exposed toforeign proteins ((antigens) produce in their blood and tissue fluidscertain soluble substances (antibodies). Such foreign proteins can besupplied by a microbiological or viral invader, in which case theantibodies serve a protective function. However, the presence of amicrobiological or viral infection is not essential to the formation ofantibodies. Any protein which is not normally present in a given animalcan, when introduced into such an animal under the proper conditions,engender the formation of antibodies. All immunological testingprocedures are based upon this so-called antigen-antibody reactionthrough the use of known diagnostic materials, either an antigen or anantibody, to determine the presence or absence of the correspondingantibody or antigen in the test animal.

When an antigen and its corresponding antibody come into contact underthe proper conditions, they combine to form a complex which is lesssoluble than were either of the uncombined original components. Thisinsoluble complex is discernible to the human eye in varying degrees.Certain antigens and antibodies will combine in a relatively shortperiod of time to form large particles which are macroscopicallyvisible. However, in many antigen-antibody systems the complex formsvery slowly, and the particle size is so small that certain carriersmust be employed to expedite the macroscopic visualization of thereaction. Among the carriers which have been employed are sheep andhuman erythrocytes, bacterial cells, bentonite and latex particles. Suchcarriers are usually characterized as indicators for each diagnostictest.

Each of the several immunological diagnostic procedures which isemployed in the art is characterized by a number of drawbacks ordisadvantages, among which are lack of sensitivity, lack of specificity,lack of stability, lack of avidity (speed and quality of reaction), andimproper character of the resultant test mixture to provide an easilyascertainable visual evaluation. In the case of certain antigen-antibodyreactions, the principal drawback is the lack of sensitivity andspecificity which often results in false positive reactions, therebynecessitating coincident controls to avoid any misinterpretation.Another principal drawback is the inordinate length of time required forthe tests. This time factor has in the past precluded many physiciansfrom utilizing the tests when elaborate laboratory facilities were notimmediately available.

It is an object of this invention to provide novel immunologicaldiagnostic procedures and diagnostic reagents which are characterized bya high degree of sensitivity and specificity and which can provideaccurate results in substantially faster time than previously wasthought possible. Another object of this invention is to provide noveldiagnostic procedures and reagents which can be routinely employed by amedical practitioner without the need of elaborate and costly laboratoryfacilities. Other objects and advantages, both general and specific,will appear as this specification proceeds.

This invention involves the preparation and use of a polymerized styrenelatex as a carrier for reactants (antigens or antibodies) which are usedfor diagnostic purposes. Optimum results are achieved when polystyrenelatex is employed in a particle size in the range of from 0.15 to 0.25micron. One such polystyrene latex suspension is available from MonsantoChemical Company under the trademark Lytron 615. The polymer particlesof styrene in the dispersed phase of the polystyrene latex suspensionaverage leSs than one micron in diameter (about 0.2 micron, with most ofthe particles being in the range of 0.15 to 0.25 micron) and arenegatively charged. The polymer is of high molecular weight and isproduced by polymerizing a styrene monomer in the presence of water toform lattices. Polystyrene latex is stable to repeated cycles offreezing and thawing and is infinitely dilutable in water. The materialappears to be selfsterilizing. Another acceptable polystyrene latexsuspension is obtainable from Koppers Company Inc., Pittsburgh, Pa.,under the trademark Dylex K-3l.

The polymer particles in the dispersed phase of this polystyrene latexand the appropriate reactant are combined in an aqueous bufferedsolution. Normally the pH is above 8.0, and excellent results areobtained at a pH of 8.2. A preferred buffered solution includes glycineand saline. Particularly advantageous results are obtained when thelatex reactant mixture is heated for a period of time. This heating stepseems to condition the reagent in such a manner that the appropriatediagnostic response is appreciably accelerated. A temperature of 57 C.for 15 to 30 minutes has been found to be satisfactory.

In order to facilitate visualization of the formation of particles, adye is incorporated into the diagnostic reagent. For these purposes,brilliant green, added as a sterile 1% solution in the proportion of1:500 in the final product, has been found to be satisfactory. Thelatexreactant preparation is stable for an indefinite period of time.The final step in the preparation of the diagnostic reagent is thedilution of the concentrated latex-reactant mixture with an appropriatebuifer. One volume of latexreactant mixture can be combined with about4.5 volumes of sterile glycine-saline buffer at pH 8.2 and containing 1%sodium azide to form a satisfactory reagent. Alternatively, theconcentrated latex-reactant mixture can be diluted with a butter priorto the heating step which would then be the final step.

In the ultimate reagent mixture excellent results have been obtainedwith the latex polymer present in an amount from about 0.2% to about0.5% and with the reactant present in an amount from about 0.05% toabout 0.1%. Normally, stock preparations will contain the latex polymerin more concentrated amount, for example, the latex polymer willcomprise 5% or more by volume of the preparation.

The diagnostic reagents of this invention comprise complexes ofpolystyrene latex with either antigens or antibodies or substances whichhave the immunological characteristics of antigens or antibodies. Highlyselective and satisfactory latex-antibody reagents have been preparedfor the determination and analysis of various blood plasma proteins,among which are gamma globulin, fibrinogen, serum albumin and C-reaetiveprotein. Equally satisfactory latex-antigen reagents have been preparedfor the diagnosis of rheumatoid factor in the serum of patients withrheumatoid arthritis and for the identification of the presence ofauto-antibodies which appear in the serum of individuals afllicted witha thyroid disorder called Hashimotos disease. Each of these diagnosticprocedures will be discussed hereinafter in greater detail.

There are a variety of gamma globulin abnormalities. A gammaglobulinemiais a disease characterized by extremely low levels of serum gammaglobulin, a marked deficiency of circulating antibodies and unusualsusceptibility to repeated severe bacterial infections. Major acuteinfections associated with a deficiency of gamma globulin are:pneumonia, hepatitis, pyelonephritis, septic arthritis and meningitis.An abnormally high level of gamma globulin with little or no antibodycontent is usually found in the myeloma group of malignant blooddisorders and may accompany hepatic disease. Gamma globulin deficiency,or disfunction may occur at any age in either sex. Most workers in thefield recommend, therefore, an examination for the possible existence ofone of these conditions whenever a patient shows unusual susceptibilityto repeated bacterial infections.

The tests which have been employed for the diagnosis of gammaglobulin.abnormalties are of two general groups. The first group includes theso-called screening tests which, although not definitive, are helpful inarriving at a preliminary diagnosis. The second group comprises thedefinitive tests which include electrophoresis, Coombs hemagglutinationinhibition test, preprecipitin reaction, gel diffusion andimmunoelectrophoresis. In general, the screening tests have lackedeither specificity or universal applicability. The definitive tests aremore diflicult and time consuming than the screening tests, requirespecial equipment or reagents, and demand a high degree of technicalskillfor their proper performance and interpretation.

A latex-antibody reagent has been successfully prepared and employed forthe determination of fibrinogen levels in blood. Hypofibrinogenemia, adeficiency in the fibrinogen blood level, is not uncommon. Suchabnormally low fibrinogen levels may occur during and following certainsurgical procedures and in cases of premature separation of theplacenta. In such situations, fibrinogen tive of certain abnormalstates. According to this inven-' tion it has been possible to preparewalatex-antibody reagent which can be employed for the quantitativedetermination of serum albumin.

Following the practice of this invention, it has been possible toprepare a latex-antibody reagent which can be employed either as ascreening procedure to detect the presense of C-reactive protein or as aquantitative test todeterminethe level of C-react-ive protein in anindividuals serum. C-reactive protein is an abnormal protein, which'iscommonly found in the serum of persons with active inflammatory ortissue-destroying disease. C- reactiveprotein is notnormally present inhuman serum. It is an acute phase protein, which usually occurs inpatients with myocardial infarction, active rheumatic fever, advancedmalignancy, rheumatoid arthritis, tuberculosis, pneumonia and otherinflammatory diseases. As inflammation lessens, C-reaotive proteindecreases; when the inflammatory process is suppressed, the abnormalprotein disappears'from the blood. The presence of C- reactive proteinis established by testing the patients serum against an anti-serumprepared by hyperimmunizingrabbits to C-reactive protein. By combiningthe C- reactive proteinantibody thereby obtained with latex particles, areagent has been produced which provides clearcut test results withintwo minutes or less.

A reagent for the diagnosis of rheumatoid arthritis is exemplary of thecombination of polystyrene latex and an antigen-like substance.Rheumatoid arthritis is a statistically incapaci-tating disease with aprevalence estimated to be about 2.5% among persons over 14 years ofage. Approximately 4,500,000 people in this country suffer from somemanifestation of the disease. The cause of rheumatoid arthritis is asyet unknown, and there is no specific cure. However, much has been donein the management of the disease. to minimize pain and control itscrippling effects. Exceedingly helpful in the treatment is thedifferentiation of rheumatoid arthritis from several other arthritidesand connective tissue diseases. To achieve this distinction physicianshave resorted to clinical criteria in addition to laboratory tests.

The laboratory tests for rheumatoid arthritis have been serologic testsfor the detection of an antibody-like or an agglutinating factorcommonly called the rheumatic factor (often abbreviated RAF or RF).Present evidence indicates that RF is probably a gamma globulin of highmolecular weight. its presence in rheumatoid serum has been demonstratedas early as the day of onset of acute symptoms. Testing for the presenceof RF has been based on the fact that it reacts with human gammaglobulin or some other reactant present in-Cohn Fraction II. CohnFraction II is a material obtained from human plasma or serum, using thefractionation method 6 and 9 of Cohn. The fraction is composed chiefly,although not exclusively, of gamma globulin.

In certain disease states of the thyroid gland, autoantibodies areformed in response to thyroid protein which is released from this glandduring the course of the pathology. One such thyroid disorder ischaracterized as Hashimotos disease. A diagnostic reagent has beenprepared by combining polystyrene latex and antigen ob tained fromeither normal or pathologic thyroid glands. This latex-antigen reagenthas been shown to be highly specific for the diagnosis of Hashimotosdisease. Such a diagnostic reagent is extremely useful, since surgicalthe use of the diagnostic reagent of this invention, it is now possibleto avoid unnecessary surgery.

When the polystyrene latex carriers which are employed i in the practiceof this invention are combined with a suitable reactant, it is possibleto perform a quick (a matter of seconds) immunological diagnosticdetermination. In the case of a test for the. presence of rheumatoidfactor, the diagnostic reagents of this invention have dramaticallylessened the time and work which has heretofore been. Ordinarily, 30;

required in the use of prior procedures. minutes to 2, hours incubationof a large number of test tubes has been required before a reading couldbe made. involved and time-consuming procedures described in the articleby Singer and Plotz in the December 1956- issue of the American Journalof Medicine and the article by Rheins et al., in the July 1957 issue ofthe Journal of Laboratory and Clinical Medicine.

The following examples illustrate in greater detail the preparation anduse of reagents which embody the teachings of this invention.

EXAMPLE I A polystyrene latex suspension known as Lytron 615, availablefrom Monsanto Chemical Company, is employed in. sterile form. The latexpolymer which in its commercial form ordinarily contains 50% solids istransferred aseptically to bottles of sterile distilled water to make a1:10 stock suspension.

The human gamma globulin reactantemployed is Cohn Fraction II. Thisreactant is dissolved ina glycine Reference can be made, for example, tothe,

saline buffer at pH 8.2 and containing 0.1% sodium azide to make a 1%solution which is sterilized by filtration through a bacteria-excludingfilter. Equal volumes of the 1% Cohn Fraction II solution and the 1:10stock latex suspension are mixed with sterile precautions, and thislatex-globulin mixture is heated at 57 C. for fifteen minutes. Thepreparation is then strained with brilliant green which is added as asterile 1% solution in the proportion of 1:90. The concentratedlatex-globulin mixture is then diluted with 4.5 volumes of sterileglycinesaline buffer at pH 8.2 and containing 0.1% sodium azide.

In the use of the reagent, a 1:20 dilution of the serum under test in aglycine-saline buffer diluent is employed. This dilution reduces thechances of false positive reaction which may occur as the result of theinteraction of certain components of non-rheumatoid serums with thelatex-globulin reagent. At the same time, rheumatoid serums withrheumatoid factor possess sufiicient activity to withstand at least thisamount of dilution. The test using the reagent provides positive andnegative control .serums for use in controlling the technique of runningthe test, as well as a check on the performance of the reagent anddiluent.

The technique of carrying out the test using the prepared reagentinvolves the following steps:

(l) Prepare a 1:20 dilution of the serum under test by adding one dropof serum to 1 ml. of the diluent (sterile glycine-saline buffer at pH8.2).

(2) Place one drop of diluted serum in a section of a divided glassslide.

(3) Add one drop of the test reagent, mix with an applicator ortoothpick, and spread over an area of approximately 20 x 25 mm.

(4) Prepare positive and negative controls, each with one drop of knownpositive and negative serums (diluted 1:20 as above under (1) and onedrop of the test reagent. Use separate applicators or toothpicks foreach mixture. Alternatively, commercially available controls of the samecharacter may be employed.

(5) Tilt the slide from side to side for 1 minute and observe formacroscopic clumping.

(6) Serums containing rheumatoid factor produce visible fluocculation ofthe test reagent whereas a smooth suspension is observed in a negativereaction. In positive tests, visible flocculation usually occurs in afew seconds.

(7) Recommended method of reading the test is as follows:

Negative-Smooth suspension with no visible flocculation.

Weakly reactiveVisible flocculation but with small aggregates or partialclumping.

Reactive-Visible flocculation with large aggregates and completeclumping (clear background).

Another advantage of the reagent of this invention is that it may alsobe used for the titration of rheumatoid serums. The density andsensitivity of the reagent is such that one drop added to one milliliterof a bufiier diluent gives a stable, smooth, good reading mixture andone drop added to one milliliter of a buffer diluent which containsrheumatoid factor gives visible flocculation to the titer of the serumspecimen.

The technique involved in carrying out the serum titration using thereagent involves the following steps:

(1) Serially number 10 test tubes (size approximately 12 x 75 mm.).Pipette 1.9 ml. of the diluent (sterile glycine-saline buffer at ph 8.2)into tube 1 and 1.0 ml. into the other tubes. Add 0.1 ml. of thepatients serum to tube 1, mix and transfer 1.0 ml. to tube 2. Mix andcontinue to prepare serial dilutions through tube 9, discarding 1.0 mi.from this tube (tube 10, the reagent control, contains no serum). Tubes1 to 9 represent, respectively, serum solutions of 1:20, 1:40, 1:80,1:160, 1:320, 1:640, 1:1280, 1:2560 and 1:5120.

(2) Add one drop of the test reagent to each tube. Shake the rack oftubes to insure thorough mixing and incubate at 37 C. for 15 minutes.

(3) Centrifuge tubes at 2500 rpm. for 2 minutes. Shake tubes gently todislodge buttons of sediment and observe for macroscopic clumping.

(4) The reagent control (tube 10) should form a smooth suspension,whereas flocculation will be obsenved in a positive reaction. The titerof the serum is the reciprocal of the highest dilution showing definiteflocculation.

Shelf-life tests of various reagents embodying combinations oflatex-globulin mixtures have shown them to withstand storage withoutvisible spontaneous flocculation under conditions varying from 4 C. to37 C. for at least twelve months. Further, reagents of the characterdescribed can be freeze-dried as by lyophilization (i.e., subjecting thereagent to reduced pressures at reduced temperatures, such as a vacuumof 75 microns at 40 C.) if further preservation is necessary.

The reagent of this invention has application in the shorter laboratoryprocedures which are preferred by some practitioners. A method ofpreparing a reagent suitable for testing specimens of whole blood is setforth in Example II.

EXAMPLE II A polystyrene latex of the type mentioned in Example I wasemployed and had a particle size in the range of 0.15 to 0.25 micron.The polystyrene latex was trans ferred aseptically to bottles of steriledistilled water to make a 1:10 stock suspension. Cohn Fraction II wasemployed as the human gamma globulin. This was dissolved in aglycine-saline buffer at pH 8.2 containing 0.1% sodium azide to make a1% solution which was thereafter sterilized by filtration through abacteria-excluding filter at room temperature. One part of the sterilelatex 1:10 stock suspension was mixed with two parts of the sterile 1%Cohn Fraction II solution. The mixture was heated at 57 C. for 15minutes. This latex-globulin preparation was then diluted with an equalvolume of sterile buffer solution at pH 8.2 and containing citrateanticoagulant. The buffer-anticoagulant diluent was prepared by mixingtwo parts of glycine-saline solution at pH 8.2 with one part of 2%sodium citrate solution at pH 8.2. The latex-globulin reagent was thenstained with neutral acrifiavine which was added as a sterile 1%solution in the proportion of 1:240.

In the use of the reagent whose preparation was just described, one dropof whole blood specimen obtained by finger or ear prick can be added totwo drops of the reagent on the surface of a flat glass slide. The bloodand reagent are mixed and spread over an area of about 20 x 40 mm. Theslide is then tilted back and forth While being observed for macroscopicclumping. Blood specimens containing rheumatoid factor produce visibleflocculation of the latex-globulin particles in a matter of seconds. Theagglutinated particles then accumulate at the periphery of thepreparation which aids in visualizing a positive reaction.

The reagent whose preparation was just described can also be used by acapillary tube method. In this procedure, one drop of whole bloodspecimen obtained by finger or ear prick can be added to two drops ofthe reagent on a non-absorbent surface such as clean paper. The bloodand reagent are mixed and a portion of the mixture is drawn into acapillary tube to a height of about 5 cm. The tip of the tube can thenbe forced into a small slab of modeling clay which serves to seal thecapillary and to hold the tube erect. Blood specimens containingrheumatoid factor produce within five minutes visible flocculation ofthe latex-globulin reagent, a phenomenon which is characterized by agranular appearance. The yellow stained clumps of latex-globulin reagentwhich cause the granularappearance of-a positive reaction become largerupon standing for or optimum for serum tests. False positive reactionsare prevented by using a larger amount of Cohn Fraction II solution thanthat requiredindiluted serum tests.. The.

latex-globulin reagent can be stained a bright yellow to facilitatereading of the test in the presence of red blood cells;

EXAMPLE III This example is concerned with the preparation anddiagnostic use of a reagent of this invention for the determination ofgamma globulin levels in blood.

The polystyrene latex suspension which is employed is that which wasdescribed in Example I. The antibody reactant is rabbit antibody tohuman serum gamma glob-- ulin prepared by conventional techniques. Thisreactant is dissolved in distilled water containing 0.1% sodium azide tomake a 1% solution which is sterilized by filtration through a-bacteriaexcluding filter. One volume of the reactant solution isdiluted with ,9 volumes of sterile glycine-saline butter at pH 8.2 andcontaining 0.1% sodium. azide, and to this is added one volume ofa5%stock latex suspension. This latex reactant mixture is heated at 57 C.for 30 minutes, and'the preparation is then stained with brilliantgreen, which is added as a sterile 1% solution in the proportion of1:500. This reagent will provide rapid and clear-cut reactionswithproper solutions of the serum under test to indicate a normal level,a clinically significant low level, or an abnormally high level of gammaglobulin.

The test is conducted as follows:

(1) To three small (10 x 70 mm.) test tubes pipette 2.5, 0.5 and 0.5 m1.of the glycine-saline buifer diluent for the serum under test.

(2) Prepare dilution 1 of the serium specimen by adding a drop of serumfrom the capillary pipette to the first tube. Discard that pipette.

(3) Mix the contents of the tube and with a fresh pipette transfer onedrop of dilution 1 to the second tube and leave the pipette in the firsttube.

(4) Mix the contents of the second tube containing serum dilution 2.With a fresh'capillary pipette'transfer onedrop of serum dilution 2 tothe third tube, and leavethe pipette in the second tube.

(5) Mix the contents of the third tube containing serium dilution 3. Puta fresh pipette in that tube.

(6) Using the pipettes which are now'in the respective tubes, transferone drop from each tubeto successive sections of a divided glass slide.Transfer one drop from each dropper bottle of normal human-controlserum,dilutions 1, 2 and 3 to the other sections of the glass slide.

Thethree dilutions of the normal human control-serum are prepared bytransferring one drop of a standard solution which contains 1 gram ofgamma globulin per-100 ml. to 2.5 ml. of glycine-saline'buifer, therebyforming dilution 1. One drop ofdilution 1 solution is transferred to 2.5ml. of glycine-saline buffer to form dilution 2. One dropof dilution 2solution is .then transferredto 2.5 ml. of glycine-saline buffer to formdilution 3.-

(7) Add one dropyof: the latex-anti-human gamma globulin reagent to eachserum dilution. Using separate toothpicks or wooden applicators, mixeach of the reaction mixtures on the slide and spread it over an area ofapproximately x mm.

(8) Tilt the slide slowlytrom side to sidefor two.

NNN Agammaglobulinemia, or less than about 25 PNN-Hypogammaglobulinemia,or approximately 50 to PPNNor-mal range, or approximately 600 .to 1200.

mg. PPP or NPP-Hypergammaglobulinemia, or more than EXAMPLE 'IV Thisexample is concerned with the preparation and diagnostic use of areagent whichis suitable for the detection and quantitation ofC-reactive protein.

The polystyrene latex employed in the reagent of this example isidentical to that set forth in Example III and is prepared in the samemanner. is combined with this latex preparation is rabbit antibody toC-reactive protein. This react-ant is prepared by conventionaltechniques. The latex-anti-C-reactive protein reagent is prepared in thesame manner as the reagent of Example III was prepared, the rabbitantibody to C- reactive protein replacing the rabbit antibody to humanserum gamma globulin of Example III.

The test is conducted in the following manner:

Qualitative Procedure 1) Using a capillary pipette, place one drop(approximately 0.02 ml.) of serum specimen in a section of a dividedslide.

(2) Add two drops of latex-anti-C-reactive protein reagent. the reactionmixture and spread it over the area of approximately 20 x 40 mm.

(3) Tilt the slide from side to side for one to two minutes and observe:for macroscopic clumping. Visible flocculation indicates the presenceof C-reactive protein. Serum devoid of this abnormal protein gives asmooth suspension with no visible flocculation Quantitative Procedure 1)Prepare serum dilutions in a glycin-salinebuffer diluent as hereinbeforedescribed. Serum specimens are tested at dilutions of 1:2, 1:4, 1: 8,1:16, 1:32 and 1:64.

(2) Using a capillary pipette transfer one drop of each serum dilutionto successive sections of a divided slide. The same capillary pipettecanbe used for a series of dilutions if the transfer is started with thehighest dilution and continued toward the lowest dilution.

(3) Add one drop .of latex-anti-C-reactive protein reagent to each dropof serum dilution. Usingia wooden applicator or toothpick.andstarting-with the highest serum dilution, mix each reaction mixtureand spread it over an area of about 20 x 40 mm.

(4) Tilt the slide from side to side for oneto two minutes andobserveior macroscopic agglutination.

(5) Interpretation of quantitative test: The highest serium solutionshowing visible flocculation is the C- reactive protein titer of theserum.

EXAMPLE V The example is concerned with the preparation and diagnosticuse of a reagent which is suitable for the deter- Prozones sometimes.

The reactant which With a wooden applicatoror toothpick mix.

mination of fibrinogen levels in blood. A polystyrene latex suspensionis employed in sterile form. The polymer particles of styrene in thedispersed phase average less than one micron in diameter (about 0.2micron with most of the particles being in the range 0.15 to 0.25micron). This latex polymer is transferred aseptically to bottles ofsterile distilled water to make a 1:10 stock suspension.

The antibody reactant is rabbit antibody to human plasma fibrinogen andis prepared by conventional techniques. The latex-anti-human fibrinogenreagent is prepared in the same manner as the reagent of Example III wasprepared, the rabbit antibody to human plasma fibrinogen replacing therabbit antibody to human serum gamma globulin of Example III.

The test is conducted in the following manner:

(1) Using a capillary pipette, transfer one drop of the test blood to 3ml. of a diluent (sterile glycine-saline buffer at pH 8.2). Thisprovides a 1:20 dilution of the blood under test.

(2) Place one drop of the diluted blood specimen in a section of adivided glass slide. Transfer one drop of a normal fi-brinogen controlpreparation to another section of the glass slide. This normalfibrinogen control is prepared from blood with a plasma fibrinogen levelof 300 mg. percent by diluting one drop in 3 ml. of glycinesaline bufferas is done with the blood under test.

(3) Add two drops of latex-anti-human fibrinogen reagent to the testblood and to the normal fibrinogen control, mix with an applicator ortoothpick and spread over the area of approximately 20 x 25 mm.

(4) Tilt the slide slowly from side to side 'for one minute and observefor clumping. Blood specimens with plasma fibrinogen levels of less than100 mg. percent will fail to show agglutination in this antigen-antibodysystem. Clumping in a degree comparable tothat shown by the normalfibrinogen control indicates a level in the normal range of 250400 mg.percent.

This application is a continuation-in-part of my co pending application,Serial No. 742,009, filed June 16, 1958, now abandoned.

While in the foregoing specification, a detailed description ofembodiments of the invention has been set forth for the purpose ofunderstanding, it will be apparent to those skilled in the art that manymodifications in the details thereof may be made without departing fromthe spirit and principles of the invention.

What is claimed is:

1. An immunological diagnostic reagent comprising 10 polymerized styrenelatex having a particle size in the range of about 0.15 to 0.25 micronand an immunological reactant selected from the group consisting ofspecific, known antibodies and antigens.

2. The reagent of claim 1 in which the reactant is present in an amountby weight of from about 0.05% to about 1.0%.

3. The reagent of claim 1 in which the latex is present in an amount byweight of from about 0.2% to about 5%.

4. An immunological diagnostic composition comprising polymerizedstyrene latex having a particle size of 0.15 to 0.25 micron and CohnFraction II.

5. In the performance of an immunological test based upon the well knownantigen-antibody reaction, which test comprises combining a reactantselected from the group consisting of specific, known antibodies andantigens, with a carrier and bringing the thus formed combination intointimate contact with a specimen suspected to contain the correspondingopposite reactant and observing the thus formed mixture for evidence ofthe formation of a macroscopic insoluble complex which indicates apositive reaction; the improvement which comprises employing as saidcarrier a polymerized styrene latex having a particle size of about 0.15to 0.25 micron.

6. The method of preparing an immunological diagnostic reagent comprisedof a reactant selected from the group consisting of specific, knownantibodies and antigens, and a polymerized styrene latex carrier whichcomprises forming a mixture of an aqueous solution of a polymerizedstyrene latex having a particle size of about 0.15 to 0.25 micron, anaqueous buffered solution of about pH 8 and said reactant, and thenheating the mixture at about 57 C. for about fifteen to thirty minutes.

7. The method of claim 6 in which after heating the mixture is stainedwith a dye.

References Cited in the tile of this patent Rheins: J. *Lab. and Clin.Med. :1, July 1957, pp. 113-118.

GG Test, trademark file of 689,310, reg. December 8, 1959, filed January29, 1959, first used in commerce June 2, 1958.

Gofton: Canadian Med. Assoc. 1., vol. 77, December 15, 1957, pp. 1098,1102.

Singer: Amer. 1 our. Med, vol. 21, December 1956, pp. 888-892.

Bozicevich: PSEBM 97:1, January 1958, pp. 180483.

Cox: J. of Lab. and Clin. Med. 48:2. August 1956, pp. 298, 299.

1. AN IMMUNOLOGICAL DIAGNOSTIC REAGENT COMPRISING POLYMERIZED STYRENELATEX HAVING A PARTICLE SIZE IN THE EANGE OF ABOUT 0.15 TO 0.25 MICRONAND AN IMMUNOLOGICAL REACTANT SELECTTED FROM THE GROUP CONSISTING OFSPECIFIC, KNOWN ANTIBODIES AND ANTIGENS.